Extremophile enzyme activity lab: using catalase from Pyrobaculum calidifontis to highlight temperature sensitivity and thermostable enzyme activity.

There are places on earth that are considered to possess extreme physico-chemical characteristics as they relate to life. Surprisingly, there are microbes that have adapted various strategies that enable them to form robust communities in these environments. The microbes that live in these environments, called extremophiles, are described as being thermophilic, psychrophilic, halophilic, acidophilic, alkaliphilic, barophilic, and so on. Given that extremophiles were not discovered until relatively recently due to a view point that the environments in which they inhabited were not conducive to life, it is reasonable to conclude that the concept of extremophiles may be hard to grasp for students. Herein is described a laboratory exercise adapted from laboratory exercises that use mesophilic catalase enzymes to illustrate the influence of physico-chemical parameters on enzyme activity. Catalase is an enzyme that accelerates the degradation of hydrogen peroxide to water and oxygen gas. In addition to mesophilic catalases, the catalase from Pyrobaculum calidifontis, a hyperthermophile with an optimal growth temperature of 90°C, is used to highlight the adaptation of an enzyme to an extreme environment. A visual comparison of bubble production by the hyperthermophilic and mesophilic enzymes after heating at high temperatures dramatically illustrates differences in thermostability that will likely reinforce concepts that are given in a pre-laboratory lecture that discusses not only the extremophiles themselves but also their applications in biotechnology and possible role in the field of astrobiology.

Designing a Wastewater-Based Epidemiology Study at the U.S. Air Force Academy: Using Severe Acute Respiratory Syndrome Coronavirus 2 to Test a Sentinel System for Early Disease Outbreak Detection.

INTRODUCTION: The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) in wastewater has been proposed as a sentinel surveillance epidemiological tool for detection of infectious disease at a community level and as a complementary approach to syndromic surveillance of infectious disease outbreaks. We have designed a study to test the presence and quantity of SARS-CoV2, the virus responsible for COVID19, in the wastewater treatment facility (WWTF) of the U.S. Air Force Academy. MATERIALS AND METHODS: Wastewater samples were tested in the laboratory to quantify the amount of SARS-CoV2 RNA using reverse transcription-quantitative polymerase chain reaction. Raw SARS-CoV2 viral titer in wastewater was normalized to the viral titer of a fecal marker, pepper mild mottle virus, to correct for dilutions. Temporal and spatial trends of COVID19 were analyzed. Furthermore, we compared wastewater analysis results against clinical data to assist public health decisions. RESULTS: Preliminary data suggest that wastewater analysis can provide temporal and spatial trends of COVID19. The geographically discrete WWTF at the U.S. Air Force suggests that wastewater testing is a useful approach to developing a comprehensive sentinel surveillance system. CONCLUSIONS: Together with ongoing syndromic surveillance data, this proof-of-concept study seeks to determine whether early detection of SARS-CoV2 in a closed system WWTF correlates to changes in community and clinically reported COVID19. The well-documented population served by the geographically discrete WWTF at the U.S. Air Force Academy may serve to better elucidate the adjunctive role of wastewater testing in a comprehensive surveillance system. These results may be of particular interest to the DoD and local commanders given the WWTFs under their immediate control and the information that these studies may provide in support of operational readiness through early detection of disease outbreaks.

Biotechnology Immersion Program: professional development where the participants do the preparation, teaching, and outreach to maximize learning gains.

Professional development for teachers of primary, intermediate, and secondary schools (Kindergarten to Grade 12; K-12), especially for highly technical subjects such as Microbial Biotechnology, can involve arduous and ineffective training methods prioritizing content delivery over sound pedagogical techniques. Teachers are learning complex content, techniques, and pedagogies but have little time to practice or gain experience and confidence in their newly acquired skills. The Biotechnology Immersion Program (BiP) sought to overcome this challenge by incorporating an intentional immersive experiential system into professional development; teachers learn new content, experience hands-on activities, and work through assessments in the role of a student while experienced subject matter expert faculty run the teaching and activities. Afterwards, the teachers get the opportunity to switch roles and practice teaching, running, and managing the same learning activities that they just experienced. The faculty experts are available to mentor, guide, and direct the teachers as they try out teaching and implementing novel biotechnology classroom activities. BiP focused on three critical aspects of successful professional development: time, personal experience, and connection. This mentored teaching and implementation practice system provided a robust professional development platform, where educators felt prepared and confident to run new biotechnology lab activities in their own classrooms.

Phylodynamics of a regional SARS-CoV-2 rapid spreading event in Colorado in late 2020.

Since the initial reported discovery of SARS-CoV-2 in late 2019, genomic surveillance has been an important tool to understand its transmission and evolution. Here, we sought to describe the underlying regional phylodynamics before and during a rapid spreading event that was documented by surveillance protocols of the United States Air Force Academy (USAFA) in late October-November of 2020. We used replicate long-read sequencing on Colorado SARS-CoV-2 genomes collected July through November 2020 at the University of Colorado Anschutz Medical campus in Aurora and the United States Air Force Academy in Colorado Springs. Replicate sequencing allowed rigorous validation of variation and placement in a phylogenetic relatedness network. We focus on describing the phylodynamics of a lineage that likely originated in the local Colorado Springs community and expanded rapidly over the course of two months in an outbreak within the well-controlled environment of the United States Air Force Academy. Divergence estimates from sampling dates indicate that the SARS-CoV-2 lineage associated with this rapid expansion event originated in late October 2020. These results are in agreement with transmission pathways inferred by the United States Air Force Academy, and provide a window into the evolutionary process and transmission dynamics of a potentially dangerous but ultimately contained variant.

Sindbis Virus Replication Reduces Dependence on Mitochondrial Metabolism During Infection.

Alphaviruses are single stranded, positive sense RNA viruses that are often transmitted through mosquito vectors. With the increasing spread of mosquito populations throughout the world, these arboviruses represent a significant global health concern. Viruses such as Sindbis Virus (SINV), Chikungunya Virus (CHIKV) and Equine Encephalitis Viruses (EEV) are all alphaviruses. As viruses, these pathogens are dependent on the host cell environment for successful viral replication. It has been observed that viruses manipulate cellular metabolism and mitochondrial shape, activity, and dynamics to favor viral infection. This report looked to understand the metabolic changes present during Sindbis virus infection of hamster and human kidney cells. Cells were infected with increasing levels of SINV and at 24 hours post infection the mitochondria morphology was assessed with staining and mitochondrial activity was measured with a real-time Seahorse Bioanalyzer. The relative amount of mitochondrial staining intensity decreased with Sindbis virus infected cells. Both oxygen consumption rate and ATP production were decreased during SINV infection while non-mitochondrial respiration and extracellular acidification rate increased during infection. Collectively, the data indicates that SINV primarily utilizes non-mitochondrial metabolism to support viral infection within the first 24 hours. This understanding of viral preference for host cell metabolism may provide critical targets for antiviral therapies and help further define the nature of alphavirus infection.

Engineering and characterization of dehalogenase enzymes from Delftia acidovorans in bioremediation of perfluorinated compounds.

Per- and Polyfluorinated alkyl substances (PFAS) are a broad class of synthetic compounds that have fluorine substituted for hydrogen in several or all locations and are globally categorized as PFCs (perfluorochemicals; commonly called fluorinated chemicals). These compounds have unique chemical and physical properties that enable their use in non-stick surfaces, fire-fighting efforts, and as slick coatings. However, recent concerns over the health effects of such compounds, specifically perfluorooctanoic acid and perfluorooctane sulfonic acid (PFOA, PFOS; PFOA/S), have led to increased attention and research by the global community into degradation methods. In this study, soil samples from PFAS-contamination sites were cultured and screened for microbes with PFOA/S degradation potential, which led to the identification of Delftia acidovorans. It was found that D. acidovorans isolated from PFAS-contaminated soils was capable of growth in minimal media with PFOA as a sole carbon resource, and an observable fluoride concentration increase was observed when cells were exposed to PFOA. This suggests potential activity of a dehalogenase enzyme that may be of use in PFOA or PFAS microbial remediation efforts. Several associated haloacid dehalogenases have been identified in the D. acidovorans genome and have been engineered for expression in Escherichia coli for rapid production and purification. These enzymes have shown potential for enzymatic defluorination, a significant step in biological degradation and removal of PFOA/S from the environment. We hypothesize that bioremediation of PFAS using naturally occurring microbial degradation pathways may represent a novel approach to remove PFAS contamination.

Campus Reset: Dynamic Planning and Response to SARS-CoV-2 Infections at the US Air Force Academy.

Predominantly asymptomatic infections, such as those for SARS-CoV-2, require robust surveillance testing to identify people who are unknowingly spreading the virus. The US Air Force Academy returned to in-person classes for more than 4000 cadets aged 18-26 years during the fall 2020 semester to meet graduation and leadership training requirements. To enable this sustained cadet footprint, the institution developed a dynamic SARS-CoV-2 response plan using near-real-time data to inform decisions and trigger policies. A surveillance testing program based on mathematical modeling and a policy-driven campus reset option provided a scaled approach to react to SARS-CoV-2 conditions. This program adequately controlled the spread of the virus for the first 2 months of the academic semester but failed to predict or initially mitigate a significant outbreak in the second half of the semester. Although this approach did not completely eliminate SARS-CoV-2 infections in the population, it served as an early warning system to alert public health authorities to potential issues, which allowed timely responses while containment was still possible.

Draft Genome Sequence of the Bacterium Delftia acidovorans Strain D4B, Isolated from Soil.

Delftia acidovorans strain D4B is an aerobic bacterium within the Betaproteobacteria lineage that was isolated from soil. The genome size is 6.26 Mbp, with a G+C content of 67%. The genome encodes enzymes potentially involved in the degradation of fluorinated compounds.

Involvement of the endocannabinoid system in the inhibition of Sindbis virus replication: a preliminary study.

BACKGROUND: Sindbis virus (Alphaviridae) is a plus-strand RNA virus that is dependent on the host cell for replication. Cannabinoid (CB) receptors are found on most human cells, including virally infected cells. Activation of cannabinoid receptors has been shown to alter normal cellular physiology. This study aimed to assess how agonist (ACEA) or antagonists/inverse agonist (AM251) of the cannabinoid receptors would alter the cellular environment and impact Sindbis virus replication. METHODS: Human hepatoma (Huh7) cells were used as our model for viral replication. Cells were infected with Sindbis virus (SINV) and then treated with CB agonist (ACEA) (10 µM) or antagonist/inverse agonist (AM-251) (10 µM) and virus replication was monitored. A double subgenomic Sindbis virus containing a green fluorescent protein (GFP) reporter gene inserted into a 3' subgenomic promoter was utilized for these assays to quickly measure viral replication. GFP fluorescent cells were analyzed using flow cytometry to measure the percentage of cells expressing the viral reporter and also quantify the levels of GFP fluorescence. RESULT: Treatment of SINV-infected Huh7 cells with CB1 receptor antagonist/inverse agonist (AM251, 10 µM) resulted in a significant decrease in viral replication, while infected cells treated with a CB1 receptor agonist (ACEA, 10 µM) resulted in a significant increase of viral infection. The data indicates that activation of CB1 receptor by cannabinoids significantly influences the ability of Sindbis virus to replicate in the host cell. CONCLUSION: Blocking CB1 receptor activity with 10 µM AM251 reduced viral replication, but activating the CB1 receptor with 10 µM ACEA resulted in an increase in viral infection. These results indicate cannabinoids may significantly impact a virus replicating in human liver cells. Future confirmation with other viruses and cell lines will be performed to better understand the impact of cannabinoids on viral infections.

Investing in our nation's future military leaders' synthetic biology knowledge to understand and recognize threats and applications.

Synthetic biology encompasses some of the greatest advancements in biology. With improvements in molecular methods and techniques that allow targeted and highly efficient genome manipulation, the capabilities of engineering biology have significantly increased. These enhancements in biotechnology represent significant potential benefits and risks to the global population. It is important that future leaders are trained and understand the incredible benefits, opportunities and risks associated with synthetic biology. The US Department of Defense (DoD) has issued a technical assessment on the future opportunities of synthetic biology and has encouraged the military institutions to expand and encourage bioengineering research programs. At the US Air Force Academy (USAFA), opportunities are provided for future Air Force officers to recognize the potential and risks associated with synthetic biology by participating in the USAFA Synthetic Biology Education Program (USBEP). Cadets can enroll in synthetic biology courses to learn and master molecular biology techniques and work on independent undergraduate research projects. In addition, cadets have the opportunity to join the USAFA's International Genetically Engineered Machine (iGEM) team and compete in the international synthetic biology competition. This report includes details on how USAFA has recruited, enrolled and encouraged synthetic biology research and education among future leaders in the US Air Force.

Stearoly-CoA desaturase 1 differentiates early and advanced dengue virus infections and determines virus particle infectivity.

Positive strand RNA viruses, such as dengue virus type 2 (DENV2) expand and structurally alter ER membranes to optimize cellular communication pathways that promote viral replicative needs. These complex rearrangements require significant protein scaffolding as well as changes to the ER chemical composition to support these structures. We have previously shown that the lipid abundance and repertoire of host cells are significantly altered during infection with these viruses. Specifically, enzymes in the lipid biosynthesis pathway such as fatty acid synthase (FAS) are recruited to viral replication sites by interaction with viral proteins and displayed enhanced activities during infection. We have now identified that events downstream of FAS (fatty acid desaturation) are critical for virus replication. In this study we screened enzymes in the unsaturated fatty acid (UFA) biosynthetic pathway and found that the rate-limiting enzyme in monounsaturated fatty acid biosynthesis, stearoyl-CoA desaturase 1 (SCD1), is indispensable for DENV2 replication. The enzymatic activity of SCD1, was required for viral genome replication and particle release, and it was regulated in a time-dependent manner with a stringent requirement early during viral infection. As infection progressed, SCD1 protein expression levels were inversely correlated with the concentration of viral dsRNA in the cell. This modulation of SCD1, coinciding with the stage of viral replication, highlighted its function as a trigger of early infection and an enzyme that controlled alternate lipid requirements during early versus advanced infections. Loss of function of this enzyme disrupted structural alterations of assembled viral particles rendering them non-infectious and immature and defective in viral entry. This study identifies the complex involvement of SCD1 in DENV2 infection and demonstrates that these viruses alter ER lipid composition to increase infectivity of the virus particles.

Microbial Murders Crime Scene Investigation: An Active Team-Based Learning Project that Enhances Student Enthusiasm and Comprehension of Clinical Microbial Pathogens.

Microbial disease knowledge is a critical component of microbiology courses and is beneficial for many students' future careers. Microbiology courses traditionally cover core concepts through lectures and labs, but specific instruction on microbial diseases varies greatly depending on the instructor and course. A common project involves students researching and presenting a disease to the class. This method alone is not very effective, and course evaluations have consistently indicated that students felt they lacked adequate disease knowledge; therefore, a more hands-on and interactive disease project was developed called Microbial Murders. For this team-based project, a group of students chooses a pathogen, researches the disease, creates a "mugshot" of the pathogen, and develops a corresponding "crime scene," where a hypothetical patient has died from the microbe. Each group gives a presentation introducing the microbial pathogen, signs/symptoms, treatments, and overall characteristics. The students then visit each other's crime scenes to match the pathogen with the correct crime scene by critically thinking through the clues. This project has shown remarkable success. Surveys indicate that 73% of students thought the project helped them understand the material and 84% said it was worth their time. Student participation, excitement, understanding, and application of microbial disease knowledge have increased and are evident through an increase in course evaluations and in student assessment scores. This project is easy to implement and can be used in a wide variety of biology, microbiology, or health classes for any level (middle school through college).

A novel system for visualizing alphavirus assembly.

Alphaviruses are small, enveloped RNA viruses that form infectious particles by budding through the cellular plasma membrane. To help visualize and understand the intracellular assembly of alphavirus virions we have developed a bimolecular fluorescence complementation-based system (BiFC) that allows visualization of capsid and E2 subcellular localization and association in live cells. In this system, N- or C-terminal Venus fluorescent protein fragments (VN- and VC-) are fused to the N-terminus of the capsid protein on the Sindbis virus structural polyprotein, which results in the formation of fluorescent capsid-like structures in the absence of viral genomes that associate with the plasma membrane of cells. Mutation of the capsid autoprotease active site blocks structural polyprotein processing and alters the subcellular distribution of capsid fluorescence. Incorporating mCherry into the extracellular domain of the E2 glycoprotein allows the visualization of E2 glycoprotein localization and showed a close association of the E2 and capsid proteins at the plasma membrane as expected. These results suggest that this system is a useful new tool to study alphavirus assembly in live cells and may be useful in identifying molecules that inhibit alphavirus virion formation.

Benzothiazole and Pyrrolone Flavivirus Inhibitors Targeting the Viral Helicase.

The flavivirus nonstructural protein 3 (NS3) is a protease and helicase, and on the basis of its similarity to its homologue encoded by the hepatitis C virus (HCV), the flavivirus NS3 might be a promising drug target. Few flavivirus helicase inhibitors have been reported, in part, because few specific inhibitors have been identified when nucleic acid unwinding assays have been used to screen for helicase inhibitors. To explore the possibility that compounds inhibiting NS3-catalyzed ATP hydrolysis might function as antivirals even if they do not inhibit RNA unwinding in vitro, we designed a robust dengue virus (DENV) NS3 ATPase assay suitable for high-throughput screening. Members of two classes of inhibitory compounds were further tested in DENV helicase-catalyzed RNA unwinding assays, assays monitoring HCV helicase action, subgenomic DENV replicon assays, and cell viability assays and for their ability to inhibit West Nile virus (Kunjin subtype) replication in cells. The first class contained analogues of NIH molecular probe ML283, a benzothiazole oligomer derived from the dye primuline, and they also inhibited HCV helicase and DENV NS3-catalyzed RNA unwinding. The most intriguing ML283 analogue inhibited DENV NS3 with an IC50 value of 500 nM and was active against the DENV replicon. The second class contained specific DENV ATPase inhibitors that did not inhibit DENV RNA unwinding or reactions catalyzed by HCV helicase. Members of this class contained a 4-hydroxy-3-(5-methylfuran-2-carbonyl)-2H-pyrrol-5-one scaffold, and about 20 µM of the most potent pyrrolone inhibited both DENV replicons and West Nile virus replication in cells by 50%.

Murine Efficacy and Pharmacokinetic Evaluation of the Flaviviral NS5 Capping Enzyme 2-Thioxothiazolidin-4-One Inhibitor BG-323.

Arthropod-borne flavivirus infection continues to cause significant morbidity and mortality worldwide. Identification of drug targets and novel antiflaviviral compounds to treat these diseases has become a global health imperative. A previous screen of 235,456 commercially available small molecules identified the 2-thioxothiazolidin-4-one family of compounds as inhibitors of the flaviviral NS5 capping enzyme, a promising target for antiviral drug development. Rational drug design methodologies enabled identification of lead compound BG-323 from this series. We have shown previously that BG-323 potently inhibits NS5 capping enzyme activity, displays antiviral effects in dengue virus replicon assays and inhibits growth of West Nile and yellow fever viruses with low cytotoxicity in vitro. In this study we further characterized BG-323's antiviral activity in vitro and in vivo. We found that BG-323 was able to reduce replication of WNV (NY99) and Powassan viruses in culture, and we were unable to force resistance into WNV (Kunjin) in long-term culture experiments. We then evaluated the antiviral activity of BG-323 in a murine model. Mice were challenged with WNV NY99 and administered BG-323 or mock by IP inoculation immediately post challenge and twice daily thereafter. Mice were bled and viremia was quantified on day three. No significant differences in viremia were observed between BG-323-treated and control groups and clinical scores indicated both BG-323-treated and control mice developed signs of illness on approximately the same day post challenge. To determine whether differences in in vitro and in vivo efficacy were due to unfavorable pharmacokinetic properties of BG-323, we conducted a pharmacokinetic evaluation of this small molecule. Insights from pharmacokinetic studies indicate that BG-323 is cell permeable, has a low efflux ratio and does not significantly inhibit two common cytochrome P450 (CYP P450) isoforms thus suggesting this molecule may be less likely to cause adverse drug interactions. However, the T1/2 of BG-323 was suboptimal and the percent of drug bound to plasma binding proteins was high. Future studies with BG-323 will be aimed at increasing the T1/2 and determining strategies for mitigating the effects of high plasma protein binding, which likely contribute to low in vivo efficacy.

Subgenomic reporter RNA system for detection of alphavirus infection in mosquitoes.

Current methods for detecting real-time alphavirus (Family Togaviridae) infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes.

Infectious alphavirus production from a simple plasmid transfection+.

We have developed a new method for producing infectious double subgenomic alphaviruses from plasmids transfected into mammalian cells. A double subgenomic Sindbis virus (TE3'2J) was transcribed from a cytomegalovirus PolII promoter, which results in the production of infectious virus. Transfection of as little as 125 ng of plasmid is able to produce 1 × 10(8) plaque forming units/ml (PFU/ml) of infectious virus 48 hours post-transfection. This system represents a more efficient method for producing recombinant Sindbis viruses.